CN118460751A - Primer combination for detecting drug resistance related locus of mycobacterium tuberculosis based on CRISPR-Cas14a and application thereof - Google Patents
Primer combination for detecting drug resistance related locus of mycobacterium tuberculosis based on CRISPR-Cas14a and application thereof Download PDFInfo
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Abstract
The invention discloses a primer combination for detecting a drug resistance related site of mycobacterium tuberculosis based on CRISPR-Cas14a and application thereof, wherein the primer combination for detecting the drug resistance related site of mycobacterium tuberculosis based on CRISPR-Cas14a comprises a gRNA transcription primer combination which is designed aiming at common mutation sites of rifampicin resistance and isoniazid resistance, and the gRNA transcription primer combination can be used for amplifying a gRNA transcription DNA template, and target specificity gRNA required by detection is obtained through in vitro transcription, and the drug resistance related mutation site of mycobacterium tuberculosis can be specifically identified and identified in the subsequent application process, so that the detection of the drug resistance of mycobacterium tuberculosis can be rapidly and accurately realized, and the method has good specificity, high sensitivity, simple method and easy popularization.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection and genotyping of biological medicines, in particular to a primer combination for detecting drug resistance related sites of mycobacterium tuberculosis based on CRISPR-Cas14a and application thereof.
Background
Currently, the success rate of treatment for drug-resistant tuberculosis (DR-TB) worldwide is 60%, which is still low. Of the antitubercular therapeutic drugs, rifampicin and isoniazid are the two most effective first-line antitubercular drugs, and resistance to these two drugs is the most alarming. At present, the cure rate of drug-resistant tuberculosis, especially multi-drug-resistant tuberculosis patients is low, the death rate of the patients is high, and serious harm is caused to human health. Therefore, it is important to diagnose tuberculosis and drug resistance accurately and timely in order to initiate proper treatment and effectively control the disease while preventing its further spread. The current diagnosis technology of drug-resistant tuberculosis mainly comprises a bacteriological detection method and a molecular biological detection method. The bacteriological diagnosis method is firstly based on the positive result of tubercle bacillus culture, and judges the drug resistance according to the growth and metabolism condition of tubercle bacillus. At present, the traditional drug sensitive test is still a gold standard for diagnosing drug resistant tuberculosis. However, the traditional solid drug susceptibility test usually needs 3 months to obtain drug susceptibility results due to slow growth speed of tubercle bacillus, and the treatment of patients with drug resistance tuberculosis is easy to delay.
In recent years, rapid development of molecular biological diagnostic techniques has provided an important means for rapid diagnosis of tuberculosis and drug-resistant tuberculosis. The detection method based on the molecular biology foundation can save the process of culturing bacteria, thus greatly shortening the diagnosis time of tuberculosis and drug-resistant tuberculosis. The currently popular molecular biology detection methods mainly include GeneXpert MTB/RIF, linear probe technology (MTBDR plus), high resolution melting curve technology (High Resolution Melting, HRM), gene chip technology and whole genome sequencing (Whole Genome Sequencing, WGS). The GeneXpert MTB/RIF is developed by Cepheid corporation in the United states, is the most widely used rifampicin drug-resistant molecule detection method at present, has the advantages of very high automation degree, simple operation, short time consumption, less cross pollution, safety to environment and laboratory staff, and is a rifampicin drug-resistant detection technology recommended by the world health organization, but the detection system can only detect the rifampicin drug-resistant condition, and the technical equipment and related reagents belong to foreign import and have high cost. The linear probe technology (MTBDR plus) can detect the sites of rifampicin, isoniazid and multi-drug resistance, provides a new means for drug resistance detection, and makes up the defect that the GeneXpert MTB/RIF technology can only detect the drug resistance of rifampicin, but the technology needs to extract DNA in the operation process, has the degree of automation inferior to that of GeneXpert Mtb/RIF, and is easy to generate false negative. The high-resolution dissolution curve method (High Resolution Melting, HRM) is a method for rapidly detecting MDR-TB by combining real-time PCR and HRM by designing specific primers aiming at genes with higher drug resistance mutation frequency, and has the advantages of visual result, easy judgment, higher sensitivity, low consumption, 2-3 days and certain false positive. Gene chip technology and Whole Genome Sequencing (WGS) can detect gene mutation of a plurality of drug-resistant sites, and the accuracy is high, but the cost is relatively high, and the method is currently applied to scientific research. Although molecular biological detection technology can rapidly diagnose the drug resistance of tubercle bacillus, the current methods all require expensive instruments, which is not beneficial to popularization and application of primary hospitals. The current molecular biological detection methods mainly detect tuberculosis drug resistance based on the principle of nucleic acid or probe hybridization, and are limited by resolution, and the methods have certain limitations in specific SNP identification.
CRISPR-Cas12a and CRISPR-Cas13a are widely used for nucleic acid detection, but their use in bacterial resistance detection is rarely reported in the literature. This is because CRISPR-Cas12a and CRISPR-Cas13a require PAM motifs for substrate recognition and achieving single base resolution without introducing additional mutant bases in the gRNA is challenging.
Disclosure of Invention
The invention mainly aims to provide a primer combination for detecting a drug resistance related locus of mycobacterium tuberculosis and application thereof, and aims to solve the problems that the existing method for detecting the drug resistance of mycobacterium tuberculosis either needs expensive instruments, is unfavorable for popularization and application of primary hospitals or is limited by base resolution and has certain limitation.
To achieve the above object, the present invention provides a gRNA transcription primer combination for detecting a drug resistance related site of mycobacterium tuberculosis based on CRISPR-Cas14a, the primer combination for detecting a drug resistance related site of mycobacterium tuberculosis based on CRISPR-Cas14a comprising a gRNA transcription primer combination comprising:
wild type rpoB-1349C of the site 1349 of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1349C is shown as SEQ ID NO. 7;
A mutant rpoB-1349T at the 1349 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1349T is shown as SEQ ID NO. 8;
A mutant rpoB-1349G of the rpoB gene 1349 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1349G is shown as SEQ ID NO. 9;
wild type rpoB-1333C at 1333 site of rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1333C is shown as SEQ ID NO. 10;
A mutant rpoB-1333T of the 1333 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1333T is shown as SEQ ID NO. 11;
a mutant rpoB-1333G of the 1333 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1333G is shown as SEQ ID NO. 12;
Wild type rpoB-1334A aiming at the 1334 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1334A is shown as SEQ ID NO. 13;
A mutant rpoB-1334T of the 1334 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1334T is shown as SEQ ID NO. 14;
a mutant rpoB-1334G of the 1334 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1334G is shown as SEQ ID NO. 15;
The sequence of the wild type rpoB-1303-1304-GA aiming at the position 1303-1304 of the rpoB gene of the mycobacterium tuberculosis is shown as SEQ ID NO. 16;
a mutant rpoB-1303T of the rpoB gene 1303 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1303T is shown in SEQ ID No. 17;
a mutant rpoB-1304T of the rpoB gene 1304 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1304TC is shown as SEQ ID NO. 18;
wild-type inhA-777G directed against the inhA gene-777 site of Mycobacterium tuberculosis, wherein the sequence of the inhA-777G is shown as SEQ ID NO. 19;
A sequence of mutant inhA-777A of inhA gene-777 site of the mycobacterium tuberculosis, wherein the inhA-777A is shown as SEQ ID NO. 20;
wild-type katG-944G directed against the site of mutation at position 944 of the KatG gene of Mycobacterium tuberculosis, wherein the sequence of the katG-944G is shown in SEQ ID NO. 21;
A mutant katG-944C directed against the katG gene 944 site of Mycobacterium tuberculosis, the sequence of the katG-944C being shown in SEQ ID NO. 22; and
A mutant KatG-944A directed against a mutation site of KatG gene 944 of said mycobacterium tuberculosis, said KatG-944A having a sequence as shown in SEQ ID No. 23.
The multiplex PCR amplification primer comprises:
rpoB-F and rpoB-R designed for the rpoB gene of Mycobacterium tuberculosis, wherein the sequence of the rpoB-F is shown as SEQ ID NO.1 and the sequence of the rpoB-R is shown as SEQ ID NO. 2;
katG-F and katG-R designed for the katG gene mutation site of the mycobacterium tuberculosis, wherein the sequence of the katG-F is shown as SEQ ID No.3 and the sequence of the katG-R is shown as SEQ ID No. 4; and
And the sequence of the inhA-F is shown as SEQ ID NO.5 and the sequence of the inhA-R is shown as SEQ ID NO. 6.
The invention provides a kit for detecting a drug-resistance related mutation site of mycobacterium tuberculosis, which comprises the multiplex PCR amplification primer and the multiplex PCR amplification primer.
In one embodiment, a detection method for detecting drug resistance of mycobacterium tuberculosis comprises the steps of:
S10, providing the kit;
S20, mixing the gRNA transcription primers of the targets in the gRNA transcription primer combination with T7-R respectively, taking synthesized gRNA skeleton DNA as a template, amplifying and applying the gRNA transcription to obtain a DNA template, taking the DNA template as a gRNA transcription template, and carrying out in vitro transcription synthesis to obtain target specific gRNA;
S30, amplifying target genes by using multiple PCR amplification primers to obtain PCR products;
S40, mixing the PCR product with a CRISPR-Cas14a detection system to obtain a fluorescent signal, and determining the type of drug resistance of the mycobacterium tuberculosis according to the intensity of the fluorescent signal, wherein the CRISPR-Cas14a detection system comprises target specific gRNA.
In one embodiment, the CRISPR-Cas14a detection system further comprises: buffer, cas14a, FQ reporter, and T7 exonuclease.
The invention also provides application of the primer combination for detecting the drug resistance related locus of the mycobacterium tuberculosis based on CRISPR-Cas14a in preparation of a diagnostic reagent, a test strip or a biosensor for detecting the drug resistance of the mycobacterium tuberculosis.
The invention discloses a primer combination for detecting a drug resistance related site of mycobacterium tuberculosis based on CRISPR-Cas14a, wherein the primer combination for detecting the drug resistance related site of mycobacterium tuberculosis based on CRISPR-Cas14a comprises a gRNA transcription primer combination which is designed aiming at common mutation sites of rifampicin resistance and isoniazid resistance, and the gRNA transcription primer combination can be used for amplifying a gRNA transcription DNA template, and target specificity gRNA required by detection is obtained through in vitro transcription, and the drug resistance related mutation site of mycobacterium tuberculosis can be specifically identified and identified in the subsequent application process, so that the detection of the drug resistance of mycobacterium tuberculosis can be rapidly and accurately realized, and the method has the advantages of good specificity, high sensitivity, simplicity and easiness in popularization.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the electrophoresis results of multiplex PCR amplification primers according to an embodiment of the present invention:
FIG. 2 is a schematic diagram of the results of a test for the effectiveness of a gRNA set according to an embodiment of the present invention.
The achievement of the objects, functional features and advantages of the present invention will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. In addition, the meaning of "and/or" as it appears throughout includes three parallel schemes, for example "A and/or B", including the A scheme, or the B scheme, or the scheme where A and B are satisfied simultaneously. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the combination of the technical solutions should be regarded as not exist and not within the protection scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
At present, the linear probe technology can detect sites of rifampicin, isoniazid and multi-drug resistance, provides a new means for drug resistance detection, and makes up the defect that the GeneXpert MTB/RIF technology can only detect the rifampicin drug resistance, but the technology needs to extract DNA in the operation process, has the degree of automation inferior to that of GeneXpert Mtb/RIF, and is easy to generate false negative. The high-resolution dissolution curve method (High Resolution Melting, HRM) is a method for rapidly detecting MDR-TB by combining real-time PCR and HRM by designing specific primers aiming at genes with higher drug resistance mutation frequency, and has the advantages of visual result, easy judgment, higher sensitivity, low consumption, 2-3 days and certain false positive. Gene chip technology and Whole Genome Sequencing (WGS) can detect gene mutation of a plurality of drug-resistant sites, and the accuracy is high, but the cost is relatively high, and the method is currently applied to scientific research. Although molecular biological detection technology can rapidly diagnose the drug resistance of tubercle bacillus, the current methods all require expensive instruments, which is not beneficial to popularization and application of primary hospitals. The current molecular biological detection methods mainly detect tuberculosis drug resistance based on the principle of nucleic acid or probe hybridization, and are limited by resolution, and the methods have certain limitations in specific SNP identification.
In view of this, the present invention proposes a primer combination for detecting a drug resistance related site of mycobacterium tuberculosis based on CRISPR-Cas14a, the primer combination for detecting a drug resistance related site of mycobacterium tuberculosis based on CRISPR-Cas14a comprising a gRNA transcribed primer combination comprising:
The sequence of the rpoB-1349C is shown in SEQ ID NO.7 aiming at the wild type rpoB-1349C at the site 1349 of the rpoB gene of the mycobacterium tuberculosis, and the sequence of the rpoB-1349C is specifically shown in the specification;
CGCCGACTGTCGGCGCTGGGGTTGCATTCCTTCATTCTTTC；
A mutant rpoB-1349T of the rpoB gene 1349 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1349T is shown in SEQ ID NO.8, and the sequence of the rpoB-1349T is specifically as follows:
CGCCGACTGTTGGCGCTGGGGTTGCATTCCTTCATTCTTTC；
a mutant rpoB-1349G of the rpoB gene 1349 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1349G is shown as SEQ ID NO.9, and the sequence of the rpoB-1349G is specifically as follows:
CGCCGACTGTGGGCGCTGGGGTTGCATTCCTTCATTCTTTC；
the sequence of the wild type rpoB-1333C aiming at the rpoB gene 1333 site of the mycobacterium tuberculosis is shown as SEQ ID NO.10, and the sequence of the rpoB-1333C is specifically as follows:
GGGGTTGACCTACAAGCGCCGTTGCATTCCTTCATTCTTTC；
a mutant rpoB-1333T of the rpoB gene 1333 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1333T is shown as SEQ ID NO.11, and the sequence of the rpoB-1333T is specifically as follows:
GGGGTTGACCGACAAGCGCCGTTGCATTCCTTCATTCTTTC；
a mutant rpoB-1333G of the rpoB gene 1333 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1333G is shown as SEQ ID NO.12, and the sequence of the rpoB-1333G is specifically as follows:
GGGGTTGACCAACAAGCGCCGTTGCATTCCTTCATTCTTTC；
The sequence of the rpoB-1334A is shown in SEQ ID NO.13 aiming at wild type rpoB-1334A at the 1334 locus of the rpoB gene of the mycobacterium tuberculosis, and the sequence of the rpoB-1334A is specifically as follows:
GGGGTTGACCCACAAGCGCCGTTGCATTCCTTCATTCTTTC；
A mutant rpoB-1334T of the rpoB gene 1334 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1334T is shown as SEQ ID NO.14, and the sequence of the rpoB-1334T is specifically as follows:
GGGGTTGACCCTCAAGCGCCGTTGCATTCCTTCATTCTTTC；
A mutant rpoB-1334G at the 1334 locus of the rpoB gene of mycobacterium tuberculosis, wherein the sequence of the rpoB-1334G is shown in SEQ ID No.15, and the sequence of the rpoB-1334G is specifically:
GGGTTGACCCGCAAGCGCCGGTTGCATTCCTTCATTCTTTC；
The sequence of the wild type rpoB-1303-1304-GA of the rpoB gene 1303-1304 of the mycobacterium tuberculosis is shown as SEQ ID NO.16, and the sequence of the rpoB-1303-1304-GA is specifically:
CCAATTCATGGACCAGAACAGTTGCATTCCTTCATTCTTTC；
A mutant rpoB-1303T of the rpoB gene 1303 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1303T is shown in SEQ ID No.17, and the sequence of the rpoB-1303T is specifically as follows:
CCAATTCATGTACCAGAACAGTTGCATTCCTTCATTCTTTC；
A mutant rpoB-1304T directed against the rpoB gene 1304 site of mycobacterium tuberculosis, wherein the sequence of the rpoB-1304TC is shown in SEQ ID No.18, and the sequence of the rpoB-1304TC is specifically:
CAATTCATGGTCCAGAACAAGTTGCATTCCTTCATTCTTTC；
Wild type inhA-777G aiming at inhA gene-777 site of mycobacterium tuberculosis, wherein the sequence of the inhA-777G is shown as SEQ ID NO.19, and the sequence of the inhA-777G is specifically as follows:
CGCGGCGAGACGATAGGTTGGTTGCATTCCTTCATTCTTTC；
The sequence of mutant inhA-777A aiming at inhA gene-777 site of mycobacterium tuberculosis, wherein the inhA-777A is shown as SEQ ID NO.20, and the sequence of inhA-777A is specifically as follows:
CGCGGCGAGATGATAGGTTGGTTGCATTCCTTCATTCTTTC；
A wild-type katG-944G directed against the site of mutation at position 944 of the KatG gene of Mycobacterium tuberculosis, wherein the sequence of the katG-944G is shown in SEQ ID NO.21, and the sequence of the katG-944G is specifically:
GCGATCACCAGCGGCATCGAGTTGCATTCCTTCATTCTTTC；
a mutant KatG-944C directed to a KatG gene 944 site of the mycobacterium tuberculosis, the sequence of the KatG-944C being shown in SEQ ID No.22, the sequence of the KatG-944C being specifically:
GCGATCACCAACGGCATCGAGTTGCATTCCTTCATTCTTTC; and
A mutant KatG-944A directed against a mutation site of KatG gene 944 of mycobacterium tuberculosis, the sequence of the KatG-944A being shown as SEQ ID No.23, the sequence of the KatG-944A being specifically:
GCGATCACCACCGGCATCGAGTTGCATTCCTTCATTCTTTC。
The primer combination for detecting the drug resistance related sites of the mycobacterium tuberculosis based on the CRISPR-Cas14a comprises a gRNA transcription primer combination which is designed aiming at common mutation sites of rifampicin resistance and isoniazid resistance, and the gRNA transcription primer combination is used for amplifying a gRNA transcription DNA template, and target specificity gRNA required by detection is obtained through in vitro transcription, so that the drug resistance of the mycobacterium tuberculosis can be detected rapidly and accurately, the specificity is good, the sensitivity is high, the method is simple, and the popularization is easy.
It should be noted that gRNA is a small RNA molecule used in CRISPR gene editing technology, and is collectively referred to as "guide RNA" and functions to direct Cas protein to a specific gene sequence. gRNA is typically composed of two parts: 1. a guide sequence complementary to a specific sequence of the target gene, thereby binding the Cas protein to the target gene; 2. functional elements, typically RNA elements used in CRISPR systems, are used to stabilize the binding of the gRNA to the Cas protein and ensure that the CRISPR/Cas system is able to accurately recognize and cleave the target gene, typically short grnas are commercially available (mature technology, low cost of synthesis) and long grnas can only be obtained by T7 polymerase in vitro transcription, since the number of gRNA bases in the present application exceeds 100nt, can only be obtained by T7 polymerase in vitro transcription.
By designing suitable gRNA primers, researchers can edit specific genes in the genome, such as targeted gene mutation, gene knockout or insertion of new DNA sequences, which makes CRISPR technology a powerful tool for researching gene functions, treating genetic diseases, improving crops and other fields.
In one embodiment, the invention further comprises a method for designing and preparing a gRNA primer sequence comprising the steps of:
summarizing mutation sites related to drug resistance of mycobacterium tuberculosis;
Selecting a high-frequency site according to the mutation site ratio;
designing the sequence of the gRNA transcription primer to match with the mutation base to be detected,
Specifically, common mutation sites of rifampicin resistance and isoniazid resistance in a guideline are summarized, statistics are carried out on the proportion of each drug-resistant mutation site to each total drug-resistant mutation, and the sites 1349, 1334, 1333, 1304 and 1303 of RpoB drug-resistant mutation related sites, 944 of kat drug-resistant mutation related sites and-777 of inhA drug-resistant mutation related sites with the highest proportion are selected. Multiple gRNA transcription primers were designed for each site, and target-specific gRNA transcription primer combinations were screened experimentally.
In one embodiment, the primer combination further comprises a multiplex PCR amplification primer comprising:
rpoB-F and rpoB-R designed for the rpoB gene of Mycobacterium tuberculosis, wherein the sequence of the rpoB-F is shown as SEQ ID NO.1 and the sequence of the rpoB-R is shown as SEQ ID NO. 2;
katG-F and katG-R designed for the katG gene mutation site of the mycobacterium tuberculosis, wherein the sequence of the katG-F is shown as SEQ ID No.3 and the sequence of the katG-R is shown as SEQ ID No. 4; and
And the sequence of the inhA-F is shown as SEQ ID NO.5 and the sequence of the inhA-R is shown as SEQ ID NO. 6.
Specifically, the design of the mutation site of the rpoB gene, namely the design of the rifampicin resistance determining region of the rpoB gene, comprises rpoB-F and rpoB-R, wherein the sequence of the rpoB-F is shown as SEQ ID NO.1, and specifically comprises the following steps: c a G a TCCGGGTCGGCATGTC; the sequence of rpoB-R is shown as SEQ ID NO.2, and specifically comprises the following steps: AGACCGATGTTGGGCCCCTC;
KatG-F and KatG-R designed for the katG gene mutation site of the mycobacterium tuberculosis, wherein the sequence of the KatG-F is shown as SEQ ID NO.3, specifically:
C*T*C*C*GCTGGAGCAGATGGGCTT；
the sequence of KatG-R is shown as SEQ ID NO.4, and specifically comprises the following steps:
TCCCACTCGTAGCCGTACAGGA；
And (3) designing inhA-F and inhA-R aiming at the inhA gene mutation site of the mycobacterium tuberculosis, wherein the sequence of the inhA-F is shown in SEQ ID NO.5, and specifically comprises the following steps:
G*T*A*A*CCCCAGTGCGAAAGTTCC；
the sequence of inhA-R is shown in SEQ ID NO.6, and specifically comprises the following steps:
AAACAGCCCCTTTGGCGCTC。
Further, the first five bases in each of the above-mentioned upstream primers F were thio-modified (expressed) and only thio-modified single-stranded DNA remained after digestion with T7 enzyme.
In the invention, the multiplex PCR amplification primer combination is designed to amplify related genes rpoB, katG and inhA of the mycobacterium tuberculosis rifampicin and isoniazid drug resistance simultaneously, so that the PCR steps are reduced, and the detection efficiency is improved.
The invention provides a kit for detecting drug resistance related sites of mycobacterium tuberculosis, which comprises the multiplex PCR amplification primer and the gRNA transcription primer, and the method can be used without expensive detection equipment, has high speed, is very suitable for large-scale popularization and application, and provides a reliable tool for rapid diagnosis of drug resistance tuberculosis.
In one embodiment, a detection method for detecting drug resistance of mycobacterium tuberculosis comprises the steps of:
s10, providing a multiplex PCR amplification primer as described above and a gRNA transcription primer combination as described above;
S20, mixing the gRNA transcription primers of the targets in the gRNA transcription primer combination with T7-R respectively, taking synthesized gRNA skeleton DNA as a template, amplifying and applying the gRNA transcription to obtain a DNA template, taking the DNA template as a gRNA transcription template, and carrying out in vitro transcription synthesis to obtain target specific gRNA;
S30, amplifying target genes by using multiple PCR amplification primers to obtain PCR products;
S40, mixing the PCR product with a CRISPR-Cas14a detection system to obtain a fluorescent signal, and determining the type of drug resistance of the mycobacterium tuberculosis according to the intensity of the fluorescent signal, wherein the CRISPR-Cas14a detection system comprises target specific gRNA obtained through in vitro transcription.
The detection method establishes a CRISPR-Cas14a MTB RIF/INH technical platform, obtains target specificity gRNA required by detection through in vitro transcription of the gRNA transcription primer combination, can guide Cas14a nuclease to identify target sites complementary with gRNA, thereby activating the nonspecific nucleic acid cutting function of Cas14a, cutting a fluorescent reporter probe to generate fluorescent signals, and enabling the nonspecific sites not to be identified, so that the nonspecific nucleic acid cutting function of Cas14a cannot be activated, and the fluorescent reporter probe is not cut or weakly cut to generate weak fluorescent signals, and can specifically identify and identify drug resistance related mutation sites of mycobacterium tuberculosis through the intensity of the fluorescent signals, so that the detection of drug resistance of mycobacterium tuberculosis can be rapidly and accurately realized, and the detection method has the advantages of good specificity, high sensitivity, simplicity and easiness in popularization.
Therefore, the dual-joint detection system for the rifampicin-resistant tuberculosis and isoniazid-resistant tuberculosis is realized, the detection steps are simple and quick, and the popularization is easy.
When needed, the gRNA transcription primer combination is designed aiming at common mutation sites of rifampicin resistance and isoniazid resistance, the gRNA transcription primer is used for amplifying a gRNA transcription DNA template, target specificity gRNA required by detection is obtained through in vitro transcription, and in the subsequent application process, the Cas14a nuclease in a CRISPR-Cas14a detection system can be guided to recognize a target site complementary with the gRNA, so that a non-specific nucleic acid cutting function of Cas14a is activated, thereby cutting a fluorescent report probe and further generating a fluorescent signal, and the non-target site cannot be recognized, so that the non-specific nucleic acid cutting function of Cas14a cannot be activated, thereby not cutting or only weakly cutting the fluorescent report probe and further only generating weak fluorescent signal, and the drug resistance related mutation site of mycobacterium tuberculosis can be specifically recognized and identified through the intensity of the fluorescent signal, thereby rapidly and accurately detecting the drug resistance of mycobacterium tuberculosis.
In step S40, the step of determining the type of drug resistance of mycobacterium tuberculosis based on the plurality of fluorescence signals includes: 1) When a fluorescent signal is generated in the targeting wild-type CRISPR reaction chamber and the corresponding targeting mutant CRISPR reaction chamber has no fluorescent signal, the site is shown to have no mutation; 2) When a fluorescent signal is generated by a CRISPR reaction chamber targeting a wild type, and a fluorescent signal is generated by a corresponding CRISPR reaction chamber targeting a mutant type, the fluorescent signal indicates that the detected substrate contains genotypes of both the wild type and the mutant type; 3) When no fluorescent signal is generated in the targeting wild-type CRISPR reaction chamber, and a fluorescent signal is generated in the corresponding targeting mutant CRISPR reaction chamber, the position is shown to have mutation; 4) When no fluorescence signal is generated in the targeting wild-type CRISPR reaction chamber, and no fluorescence signal is generated in the corresponding targeting mutant CRISPR reaction chamber, the mutation of the non-drug-resistant related site is indicated.
In one embodiment, the CRISPR-Cas14a detection system comprises: buffer, gRNA, cas14a, FQ reporter and T7 exonuclease.
It should be noted that, the CRISPR family member Cas14a does not depend on PAM motif to carry out substrate recognition, so that real single base recognition can be realized, and therefore, the method is very suitable for detecting drug resistance caused by single base mutation, and by establishing CRISPR-Cas14a MTB RIF/INH technical platform, a dual detection system of rifampicin-resistant tuberculosis and isoniazid-resistant tuberculosis is realized, so that the CRISPR-Cas14a MTB RIF/INH technology can accurately detect gene mutation related to rifampicin and isoniazid, and meanwhile, the CRISPR-Cas14a MTB RIF/INH technology does not need to depend on expensive detection equipment to realize rifampicin-resistant and isoniazid tuberculosis detection, and has the advantages of low cost, high speed, and is very suitable for large-scale popularization and application, and provides a reliable tool for rapid diagnosis of drug-resistant tuberculosis.
The invention also provides application of the primer combination for detecting the drug resistance related locus of the mycobacterium tuberculosis based on CRISPR-Cas14a in preparation of a diagnostic reagent, a test strip or a biosensor for detecting the drug resistance of the mycobacterium tuberculosis, and various reagents or reagent strips can be prepared from the primer combination for detecting the drug resistance of the mycobacterium tuberculosis.
The following technical solutions of the present invention will be described in further detail with reference to specific examples and drawings, and it should be understood that the following examples are only for explaining the present invention and are not intended to limit the present invention.
Example 1: design and screening of multiplex PCR amplification primers
In the invention, for the genes rpoB, katG and inhA promoter related to rifampicin resistance and isoniazid resistance, PRIMER PREMIER V5.0.0 software is used for designing multiplex PCR amplification primers and simultaneously amplifying rpoB, katG and inhA promoter (specifically shown in table 1), as shown in figure 1: in the agarose gel electrophoresis detection multiplex PCR amplification effect diagram, 1-9 are tuberculosis strains separated from nine tuberculosis patients respectively, 10 is a negative control, three strips of RpoB, katG and inhA are clearly amplified by the designed multiplex PCR amplification primers, and no impurity strip exists, so that the designed multiplex PCR amplification primers are successful and can be applied to subsequent experiments.
TABLE 1 multiplex PCR amplification primers
Example 2: gRNA primer sequence design, preparation and effectiveness test
(1) GRNA primer sequence design
According to the invention, common mutation sites of rifampicin resistance and isoniazid resistance in the patent and guideline are summarized, statistics is carried out on the proportion of each drug-resistant mutation site to each total drug-resistant mutation, and the gRNA primer design is carried out by selecting KatG-R50L, katG-R50W, H445Y, H445D, H445L, H445R, D435Y, D435V, katG-F15T, inhA _C-15T with the highest proportion.
GRNA primer sequence design principle: the length of the sequence matched with the target of the gRNA primer is 20bp, and the mutation base to be detected is matched with the 9 th-10 th base of the gRNA primer. Primer sequences for grnas are shown in table 2 below: (the underlined sequence is the one that matches the detection target).
TABLE 2 sequences of each gRNA primer in the gRNA transcription primer combinations
(2) Preparation of gRNA
Amplifying a DNA transcription template of the gRNA from a PUC19 vector containing a gRNA skeleton sequence (SEQ ID NO. 24) of a T7 promoter by using a gRNA transcription (SEQ ID NO. 7-23) primer and T7-R (SEQ ID NO. 25) aiming at each drug-resistant detection site through PCR, using an amplified PCR product as the DNA template, carrying out in vitro transcription at 37 ℃ by using a T7 rapid high-yield RNA synthesis kit to obtain target-specific gRNA, and purifying by using an RNA purification kit (NEB) to obtain purified gRNA, and storing at-70 ℃ for later use, wherein the gRNA skeleton sequence is shown as SEQ ID NO.24 and comprises the following specific sequences:
GTTGCATTCCTTCATTCTTTCAAATGAATTTGTTTCGAGGGTTACTTTCCGAAGAAAGCACTTCTCGACATTAGGCTGATGCAAGCAGCCCACCTTCACTCAAGTTCTAATCCCCTAAGGGACAGCTTTTGGTGAAGCGGTTCTCCACTTTATCAGTGAAccctatagtgagtcgtattagaattc;
The sequence of T7-R is shown as SEQ ID NO.25, and the specific sequence is as follows:
GAATTCTAATACGACTCACTATAGGGTTCACTGATAAAGTGG。
(3) Testing of gRNA validity
The CRISPR-Cas14a detection system comprises: 100nM Cas14a, 100nM target-specific gRNA, 100nM FQ reporter gene, 100nM nM substrate DNA, nuclease assay buffer NEbuffer4.0, total reaction volume of 20 μl. Reactions were monitored at 37℃for the indicated times using a fluorescence plate reader (VarioskanFlash, thermoFisherScientific, MA, USA). Fluorescence measurement using an excitation wavelength of 492nm and an emission wavelength of 520nm, the detection results are shown in FIG. 2: in the figure, the ordinate represents fluorescence intensity, the abscissa represents detected wild type and mutant target ssDNA, and fluorescence intensity is detected after CRISPR reaction for 1 hour. t-test was used to evaluate the significant differences. (n=3 technical replicates; bars represent mean ± SEM, p <0.05, p <0.01, p <0.001, p < 0.0001), the results indicate: in the CRISPR reaction system, the strongest fluorescence signal can be excited when the gRNA detection site and the site sequence of the substrate ssDNA are all the time, and the fluorescence signal is weaker when the detection site of the gRNA is inconsistent with the site of the substrate ssDNA. Therefore, the screened gRNA can specifically identify own target sites and cause strong fluorescent signals, so that the designed gRNA can specifically identify and identify the drug-resistance related mutation sites of the mycobacterium tuberculosis, and can be used for drug-resistance detection of the rifampicin and isoniazid of the mycobacterium tuberculosis.
Example 3: clinical samples assess the feasibility of the CRISPR-CarpoB-F4aMTBRIF/INH method
(1) Extraction of sputum sample DNA: in total, 55 sputum specimens were collected and the smear analysis result was positive. RIF and INH drug susceptibility tests were performed on culture positive patient specimens. Of the 55 samples, 8 were identified as multi-resistant, 4 showed RIF single resistance, 3 showed INH single resistance, and 41 were sensitive to both RIF and INH. DNA from each sputum sample was extracted using a commercial DNA extraction kit (not limited to company brands).
(2) Preparing target DNA: amplification was performed using the multiplex PCR amplification primers prepared in example 1, enriching target DNA, multiplex PCR amplification reaction system reference multiplexPCRKit (nuuzan, south kyo, china): 6 μl multiplex PCR amplification primer combination (final concentration of each primer 0.2 μM) and 100pg DNA template, final volume 50 μl, PCR thermocycling parameters were set as follows: initial denaturation was carried out at 95℃for 3min, at 95℃for 30s, at 60℃for 30s, at 72℃for 1min for a total of 35 cycles. The final extension step was performed at 72℃for 10 minutes. After amplification, the PCR products were analyzed using 1% agarose gel electrophoresis.
(3) CRISPR detects rifampicin and isoniazid resistance: the CRISPR-Cas 14a detection system is: in NEBuffer4.0 buffer solution, 100nM CarpoB-F4a, 100nM gRNA, 100nM FQ reporter gene, 1U T7 exonuclease (NEB company) and 2ul multiplex PCR products are composed, the total volume is 20 [ mu ] l, fluorescence values are measured after incubation for 1-2 hours at 37 ℃, and excitation wavelength of 492nM and emission wavelength of 520nM are adopted for fluorescence measurement.
(4) Drug resistance result interpretation: 1) When a fluorescent signal is generated in the targeting wild-type CRISPR reaction chamber and the corresponding targeting mutant CRISPR reaction chamber has no fluorescent signal, the site is shown to have no mutation; 2) When a fluorescent signal is generated by a CRISPR reaction chamber targeting a wild type, and a fluorescent signal is generated by a corresponding CRISPR reaction chamber targeting a mutant type, the fluorescent signal indicates that the detected substrate contains genotypes of both the wild type and the mutant type; 3) When no fluorescent signal is generated in the targeting wild-type CRISPR reaction chamber, and a fluorescent signal is generated in the corresponding targeting mutant CRISPR reaction chamber, the position is shown to have mutation; 4) When no fluorescence signal is generated in the targeting wild-type CRISPR reaction chamber, and no fluorescence signal is generated in the corresponding targeting mutant CRISPR reaction chamber, the mutation of the non-drug-resistant related site is indicated. This example relates to assessing resistance of clinical sputum samples using the CRISPR-Cas14a MTBRIF/INH method. The detection sensitivity and feasibility of the method for rifampicin and isoniazid resistance are proved by DNA extraction, target DNA preparation and CRISPR detection of sputum samples.
(5) Compared with the drug sensitivity test results, the drug resistance diagnostic performance is detected, and the results of drug resistance and sensitivity are shown in table 3:
TABLE 3 clinical samples evaluation of drug resistance diagnostic performance of CRISPR-Cas14a RIF/INH method
As can be seen from table 3: the results of the drug resistance test and the sensitivity test, and the calculated sensitivity and specificity data are given in the table, and the sensitivity calculation formula is: sensitivity = true positive number/(true positive number + false negative number) ×100%, specificity = true negative number/(true negative number + false positive number).
Based on the data in the tables, resistance to rifampicin was evaluated:
The number of true positives is 12, the number of false negatives is 0, the number of true negatives is 43, and the number of false positives is 2, so that the sensitivity result is 12/(12+2) ≡85.7%, and the Confidence Interval (CI) of 95% is (71.2% -93.9%);
The specificity of rifampicin was 43/(43+0) =100%, with 95% confidence interval at (98%, 99.8%).
Evaluation of isoniazid resistance performance based on data in the table: the sensitivity of isoniazid is 11/(11+0) =100%, and the 95% Confidence Interval (CI) is (90.8%, 99.8%) and the specificity of isoniazid is 44/(44+0) =1=100%, and the 95% Confidence Interval (CI) is (97.9% -99.7%).
From the above results, it can be concluded that: the CRISPR-Cas14aRIF/INH method has good performance in the aspects of detecting the drug sensitivity of rifampicin and isoniazid, and has high sensitivity and specificity.
The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention, but various modifications and variations will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A primer combination for detecting a mycobacterium tuberculosis drug resistance related site based on CRISPR-Cas14a, wherein the primer combination for detecting a mycobacterium tuberculosis drug resistance related site based on CRISPR-Cas14a comprises a gRNA transcription primer combination comprising:
wild type rpoB-1349C of the site 1349 of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1349C is shown as SEQ ID NO. 7;
A mutant rpoB-1349T at the 1349 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1349T is shown as SEQ ID NO. 8;
A mutant rpoB-1349G of the rpoB gene 1349 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1349G is shown as SEQ ID NO. 9;
wild type rpoB-1333C at 1333 site of rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1333C is shown as SEQ ID NO. 10;
A mutant rpoB-1333T of the 1333 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1333T is shown as SEQ ID NO. 11;
a mutant rpoB-1333G of the 1333 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1333G is shown as SEQ ID NO. 12;
Wild type rpoB-1334A aiming at the 1334 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1334A is shown as SEQ ID NO. 13;
A mutant rpoB-1334T of the 1334 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1334T is shown as SEQ ID NO. 14;
a mutant rpoB-1334G of the 1334 locus of the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1334G is shown as SEQ ID NO. 15;
The sequence of the wild type rpoB-1303-1304-GA aiming at the position 1303-1304 of the rpoB gene of the mycobacterium tuberculosis is shown as SEQ ID NO. 16;
a mutant rpoB-1303T of the rpoB gene 1303 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1303T is shown in SEQ ID No. 17;
a mutant rpoB-1304T of the rpoB gene 1304 site of the mycobacterium tuberculosis, wherein the sequence of the rpoB-1304TC is shown as SEQ ID NO. 18;
wild-type inhA-777G directed against the inhA gene-777 site of Mycobacterium tuberculosis, wherein the sequence of the inhA-777G is shown as SEQ ID NO. 19;
A sequence of mutant inhA-777A of inhA gene-777 site of the mycobacterium tuberculosis, wherein the inhA-777A is shown as SEQ ID NO. 20;
wild-type katG-944G directed against the site of mutation at position 944 of the KatG gene of Mycobacterium tuberculosis, wherein the sequence of the katG-944G is shown in SEQ ID NO. 21;
A mutant katG-944C directed against the katG gene 944 site of Mycobacterium tuberculosis, the sequence of the katG-944C being shown in SEQ ID NO. 22; and
A mutant KatG-944A directed against a mutation site of KatG gene 944 of said mycobacterium tuberculosis, said KatG-944A having a sequence as shown in SEQ ID No. 23.
2. The primer combination for detecting a mycobacterium tuberculosis drug resistance-related site based on CRISPR-Cas14a of claim 1, wherein the primer combination further comprises a multiplex PCR amplification primer comprising:
rpoB-F and rpoB-R designed for the rpoB gene of the mycobacterium tuberculosis, wherein the sequence of the rpoB-F is shown as SEQ ID NO.1, and the sequence of the rpoB-R is shown as SEQ ID NO. 2;
katG-F and katG-R designed for the katG gene mutation site of the mycobacterium tuberculosis, wherein the sequence of the katG-F is shown as SEQ ID NO.3, and the sequence of the katG-R is shown as SEQ ID NO. 4; and
And the sequence of the inhA-F is shown as SEQ ID NO.5, and the sequence of the inhA-R is shown as SEQ ID NO. 6.
3. A kit for detecting a drug resistance-associated mutation site in mycobacterium tuberculosis, comprising the gRNA transcription primer combination of claim 1 and the multiplex PCR amplification primer of claim 2.
4. A detection method for detecting a drug resistance-related mutation site of mycobacterium tuberculosis, comprising the steps of:
S10, providing the kit of claim 3;
S20, mixing the gRNA transcription primers of the targets in the gRNA transcription primer combination with T7-R respectively, taking synthesized gRNA skeleton DNA as a template, amplifying and applying the gRNA transcription to obtain a DNA template, taking the DNA template as a gRNA transcription template, and carrying out in vitro transcription synthesis to obtain target specific gRNA;
S30, amplifying target genes by using multiple PCR amplification primers to obtain PCR products;
S40, mixing the PCR product with a CRISPR-Cas14a detection system to obtain a fluorescent signal, and determining the type of drug resistance of the mycobacterium tuberculosis according to the intensity of the fluorescent signal, wherein the CRISPR-Cas14a detection system comprises target specific gRNA.
5. The method for detecting a drug resistance-associated mutation site of mycobacterium tuberculosis according to claim 4, wherein in step S40, the CRISPR-Cas14a detection system further comprises: buffer, cas14a, FQ reporter, and T7 exonuclease.
6. The use of a primer combination for detecting a mycobacterium tuberculosis drug resistance-related site based on CRISPR-Cas14a according to claim 1 in the preparation of a diagnostic reagent, test strip or biosensor for detecting mycobacterium tuberculosis drug resistance.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154527A (en) * | 2011-05-12 | 2011-08-17 | 遵义医学院附属医院 | Method for rapidly detecting multi-drug resistant tuberculosis |
| CN102559916A (en) * | 2012-02-24 | 2012-07-11 | 孙爱华 | Method for detecting multi-drug resistance of Mycobacterium tuberculosis |
| CN112725487A (en) * | 2021-02-03 | 2021-04-30 | 张国良 | Nucleic acid rapid detection kit for streptomycin drug-resistant mycobacterium tuberculosis and detection method thereof |
| CN114107300A (en) * | 2021-11-30 | 2022-03-01 | 宁夏医科大学 | CRRNA for detecting rifampicin resistance point mutation of mycobacterium tuberculosis based on CRISPR-Cas technology |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154527A (en) * | 2011-05-12 | 2011-08-17 | 遵义医学院附属医院 | Method for rapidly detecting multi-drug resistant tuberculosis |
| CN102559916A (en) * | 2012-02-24 | 2012-07-11 | 孙爱华 | Method for detecting multi-drug resistance of Mycobacterium tuberculosis |
| CN112725487A (en) * | 2021-02-03 | 2021-04-30 | 张国良 | Nucleic acid rapid detection kit for streptomycin drug-resistant mycobacterium tuberculosis and detection method thereof |
| CN114107300A (en) * | 2021-11-30 | 2022-03-01 | 宁夏医科大学 | CRRNA for detecting rifampicin resistance point mutation of mycobacterium tuberculosis based on CRISPR-Cas technology |
Non-Patent Citations (1)
| Title |
|---|
| LUCAS B. HARRINGTON等: "Programmed DNA destruction by miniature CRISPR-Cas14 enzymes", SCIENCE, vol. 362, 16 November 2018 (2018-11-16), pages 1 - 2 * |
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